One of the most important and fundamental processes in biology is the division of cells mediated by the cell cycle. This process ensures the controlled production of subsequent generations of cells with defined biological function. It is a highly regulated phenomenon and responds to a complex set of cellular signals both within the cell and from external sources. A complex network of tumor promoting and suppressing gene products are key components of this cellular signalling process. Over-expression of tumor-promoting components or the subsequent loss of the tumor-suppressing products will lead to unregulated cellular proliferation and the generation of tumors (Pardee, Science 246: 603-608, 1989).
Kinases are important cellular enzymes that perform essential cellular functions such as regulating cell division and proliferation, and also appear to play a decisive role in many disease states that are characterized by uncontrolled proliferation and differentiation of cells. These disease states encompass a variety of cell types and maladies such as cancer, atherosclerosis, restenosis and other proliferative disorders (Kris M G et al., JAMA 290 (16): 2149-58, 2003).
Cyclin-dependent kinases (CDKs) are relatively small proteins, with molecular weights ranging from 34 to 40 kDa, and contain little more than the kinase domain. CDK binds a regulatory protein called a cyclin. Without cyclin, CDK has little kinase activity; only the cyclin-CDK complex is an active kinase. CDKs phosphorylate their substrates on serines and threonines, so they are serine-threonine kinases (Morgan, D. O., Cell Division, 2:27, 2007).
The members of the cyclin-dependent kinase (CDK) family play critical regulatory roles in cell proliferation. There are currently 20 known mammalian CDKs. While CDK7-13 and 18 have been linked to transcription, only CDK1, 2, 4 and 6 show demonstrable association with the cell cycle. Unique among the mammalian CDKs, CDK7 has consolidated kinase activities, regulating both the cell cycle and transcription. In the cytosol, CDK7 exists as a heterotrimeric complex and is believed to function as a CDK1/2-activating kinase (CAK), whereby phosphorylation of conserved residues in CDK1/2 by CDK7 is required for full catalytic CDK activity and cell cycle progression (Desai et al., Mol. Cell Biol. 15, 345-350, 1995).
CDK7, which complexes with cyclin H and MAT1, phosphorylates the cell cycle CDKs in the activation of T-loop, to promote their activities (Fisher et al., Cell., August 26; 78(4):713-24, 1994). As such, it has been proposed that inhibiting CDK7 would provide a potent means of inhibiting cell cycle progression, which may be especially relevant given that there is compelling evidence from gene knockout studies in mice for lack of an absolute requirement for CDK2, CDK4 and CDK6 for the cell cycle, at least in most cell types (Malumbres et al., Nature Cell Biology, 11, 1275-1276, 2009), whilst different tumors appear to require some, but be independent of other interphase CDKs (CDK2, CDK4, CDK6). Recent genetic and biochemical studies have confirmed the importance of CDK7 for cell cycle progression (Larochelle et al., Mol Cell., March 23; 25(6):839-50. 2007; Ganuza et al., EMBO J., May 30; 31(11): 2498-510, 2012).
Cyclin-dependent kinase 7 (CDK7) activates cell cycle CDKs and is a member of the general Transcription factor II Human (TFIIH). CDK7 also plays a role in transcription and possibly in DNA repair. The trimeric Cak complex CDK7/CyclinH/MAT1 is also a component of TFIIH, the general transcription/DNA repair factor IIH (Morgan, D. O., Annu. Rev. Cell Dev. Biol. 13, 261-91, 1997). As a TFIIH subunit, CDK7 phosphorylates the CTD (Carboxy-Terminal-Domain) of the largest subunit of RNA polymerase II (pol II). The CTD of mammalian pol II consists of 52 heptad repeats with the consensus sequence 1YSPTSPS7 and the phosphorylation status of the Ser residues at positions 2 and 5 has been shown to be important in the activation of RNAP-II indicating that it is likely to have a crucial role in the function of the CTD. CDK7, which primarily phosphorylates Ser-5 (PS5) of RNAP-II at the promoter as part of transcriptional initiation (Gomes et al., Genes Dev. 2006 Mar. 1; 20(5):601-12, 2006), incontrast with CDK9, which phosphorylates both Ser-2 and Ser-5 of the CTD heptad (Pinhero et al., Eur. J. Biochem., 271, pp. 1004-1014, 2004).
In addition to CDK7, other CDKs have been reported to phosphorylate and regulate RNA pol (II) CTD. The other CDKs include, Cdk9/Cyclin T1 or T2 that constitute the active form of the positive transcription elongation factor (P-TEFb) (Peterlin and Price, Mol Cell., August 4; 23(3): 297-305, 2006) and Cdk12/Cyclin K and Cdk13/Cyclin K as the latest members of RNAPII CTD kinases (Bartkowiak et al., Genes Dev., October 15; 24(20):2303-16, 2010; Blazek et al., Genes Dev. October 15; 25(20):2158-72, 2011).
Disruption of RNAP II CTD phosphorylation has been shown to preferentially effect proteins with short half-lives, including those of the anti-apoptotic BCL-2 family. (Konig et al., Blood, 1, 4307-4312, 1997; The transcriptional non-selective cyclin-dependent kinase inhibitor flavopiridol induces apoptosis in multiple myeloma cells through transcriptional repression and down-regulation of Mc1-1; (Gojo et al., Clin. Cancer Res. 8, 3527-3538, 2002).
This suggests that the CDK7 enzyme complexes are involved in multiple functions in the cell: cell cycle control, transcription regulation and DNA repair. It is surprising to find one kinase involved in such diverse cellular processes, some of which are even mutually exclusive. It also is puzzling that multiple attempts to find cell cycle dependent changes in CDK7 kinase activity remained unsuccessful. This is unexpected since activity and phosphorylation state of its substrate, CDC2, fluctuate during the cell cycle. In fact, it is shown that cdk7 activity is required for the activation of both Cdc2/Cyclin A and Cdc2/Cyclin B complexes, and for cell division. (Larochelle, S. et al. Genes Dev 12, 370-81, 1998). Indeed, flavopiridol, a non-selective pan-CDK inhibitor that targets CTD kinases, has demonstrated efficacy for the treatment of chronic lymphocytic leukemia (CLL), but suffers from a poor toxicity profile (Lin et al., J. Clin. Oncol. 27, 6012-6018, 2009; Christian et al., Clin. Lymphoma Myeloma, 9, Suppl. 3, S179-S185, 2009).
In-vitro studies revealed substrate preferences for the different CDK7 complexes, indicating that CDK7 may form different complexes with different substrate specificity and presumably different in-vivo functions (Frit, P. et al., Biochimie 81, 27-38, 1999; Schutz, P. et al. Cell 102, 599-607, 2000).
Thus in view of the role transcriptional CDKs play in the regulation of cell cycle, there is a need of compounds to treat diseases and/or disorder associated with selective transcriptional CDKs including CDK7, CDK9, CDK12, CDK13 and CDK18; more particularly CDK7. It is, therefore, an object of this invention to provide compounds useful in the treatment and/or prevention or amelioration of such diseases and/or disorder.